Mesoporous internal immunoassay analysis model constructed based on core-shell structure magnetic hierarchical porous ZIFs and preparation method and application of mesoporous internal immunoassay analysis model

Abstract

The invention discloses a mesoporous channel internal immunoassay analysis model constructed based on core-shell structure magnetic hierarchical porous ZIFs and a preparation method and application thereof.The preparation method comprises the steps that mesoporous ZIF-8 grows outside Fe3O4 magnetite nanoparticles through a soft template oriented self-assembly and constrained growth mechanism, and a superparamagnetic core-shell structure Fe3O4-coated HMZIF-8 with Fe3O4NPs as a core and HMZIF-8 as a shell is obtained; the structure is used for constructing an in-mesoporous immunoassay analysis model. According to the immunoassay analysis model disclosed by the invention, a stable microenvironment is provided by virtue of a large mesoporous channel so as to maintain the recognition capability of a captured antibody, the sensitivity of immunoassay is remarkably improved, the content of low-concentration cTnI in serum can be accurately quantified, and the luminous quantitative detection on the cTnI in a complex biological sample is not interfered. A novel nano platform for accurately diagnosing different diseases is obtained by changing the type of a primary capture antibody of IMIA.

Description

Technical Field

[0001] The present invention relates to immunoassays, and more particularly to a mesoporous immunoassay analysis model constructed based on core-shell structured magnetic hierarchical porous ZIFs, and a preparation method and application thereof. Background Art

[0002] Sensitively quantifying low-abundance biomarkers in blood is crucial for the early screening and diagnosis of various diseases. Enzyme-linked immunosorbent assay (ELISA) is an analytical technique based on the specific recognition of antigen-antibody and efficient enzyme-catalyzed reactions (Adv. Mater. 2022, 34, 2106607). Due to its simple operation, low cost, direct reading and safety, it has been widely used in the detection of biomarker proteins, and its typical limit of detection (LOD) ranges from 0.1 ng mL -1 to 1 μg mL -1 . The low surface area of the two-dimensional (2D) plane of the microplate usually limits the density of the coated antibody, which is also one of the reasons for its relatively high LOD (Chem. Rev. 2017, 117, 9973-10042). In addition, the clinical thresholds of many protein biomarkers, especially in the early stages of diseases, are often lower than the LOD of traditional ELISA. Therefore, there is an urgent need to improve the sensitivity of current ELISA for detecting low-abundance protein biomarkers.

[0003] The emergence of microspheres in immunoassay (IA) brings new hope because this spherical carrier can overcome some of the main deficiencies of current biological detection platforms (Adv. Funct. Mater. 2024, 2409632). Compared with two-dimensional structures, the three-dimensional structure of microspheres can provide a relatively large specific surface area and higher spatial freedom. Among them, magnetic bead-based immunocapture technology stands out due to its high capture efficiency and magnetic field-assisted rapid separation and enrichment (ACS Appl. Mater. Interfaces 2023, 15, 55346 - 55357). However, the coupling process of capture antibodies or other molecules to the surface of magnetic beads is usually complex, generally requiring optimized reaction conditions and cumbersome coupling chemical reactions. At the same time, the specific surface area and fixed amount of antibodies on magnetic beads are still not satisfactory. Therefore, magnetic bead-based enzyme-linked immunosorbent assay still has certain limitations, such as insufficient recognition sensitivity for challenging bioentity molecules in cardiovascular or neurological diseases. Currently, hierarchical mesoporous zeolitic imidazolate frameworks (HMZIFs) are a class of booming crystalline materials with dual-ordered microporous and mesoporous structures that can accommodate macromolecules of various sizes, providing an optimized reaction space for ELISA. Compared with the outer surface of magnetic beads, the mesoporous channels of HMZIFs provide a larger surface area and open pore space (Adv. Funct. Mater. 2023, 33, 2215192), which enables the coverage of high-density antibodies in the mesopores, thereby improving the capture efficiency for low-abundance disease biomarkers and making in-mesopore IA possible. Therefore, it is necessary to develop an appropriate strategy to construct magnetic HMZIFs to achieve IMIA for detecting low-abundance biomarkers. Summary of the Invention

[0004] The object of the present invention is to provide an in-mesopore immunoassay analysis model constructed based on core-shell structured magnetic hierarchical porous ZIFs, its preparation method and application, so as to solve the problems of insufficient sensitivity, limited surface area and complex coupling process in the detection of low-abundance biomarkers in the prior art (such as traditional ELISA and magnetic bead technology).

[0005] To solve the above problems, the present invention adopts the following technical solutions:

[0006] According to the first aspect of the present invention, there is provided a preparation method for constructing an in-mesopore immunoassay analysis model based on core-shell structured magnetic hierarchical porous ZIFs, which includes the steps: S1, synthesizing Fe 3 O 4 NPs by solvothermal method; S2, in Fe 3 O 4The outer surface of the NPs was coated with polyvinylpyrrolidone (PVP) polymer to promote the epitaxial growth of ZIF-8 on the surface of magnetic NPs, resulting in Fe 3 O 4 @ZIF-8; S3, magnetic Fe was obtained through a soft-template-directed self-assembly and confinement growth strategy 3 O 4 @HMZIF-8, with its core being Fe 3 O 4 NPs, and HMZIF-8 as the shell layer. This unique core-shell structure enabled the construction of an immunoassay analysis model within the mesopores.

[0007] Preferably, in step S2, the outer surface of Fe 3 O 4 NPs was modified with polyvinylpyrrolidone. The hydrophilic PVP segments therein could coordinate with Zn 2+ to provide sufficient interactions, solve the phase separation problem, and ensure the self-assembly of ZIF-8 on Fe 3 O 4 NPs.

[0008] Preferably, in step S3, using PS 45 -PVP 44 -PEO 113 triblock copolymer micelles as structure-directing agents, HMZIF-8 was continuously crystallized on the surface of Fe 3 O 4 @ZIF-8 to form a mesoporous shell layer; the introduction of a hydrophobic pore swelling agent could precisely control the pore size, resulting in magnetic hierarchical porous ZIFs (Fe 3 O 4 @HMZIF-8) with a core-shell structure.

[0009] Preferably, step S1 includes: dissolving FeCl 3 ·6H 2 O (1.3 g) and sodium citrate (0.5 g) in ethylene glycol (40 mL). Then, add CH 3 COONa (1.20 g) and stir for 30 min. Transfer the mixture and seal it in a stainless-steel autoclave lined with polytetrafluoroethylene. The autoclave was heated at 200 °C for 8 h and then cooled to room temperature. Finally, wash it several times with ethanol and deionized water, and the product was dried overnight in vacuo at 60 °C.

[0010] Preferably, step S2 includes: taking 10 mg of Fe 3 O 4NPs were added to 2.4 mL of methanol containing 0.16 g of PVP and sonicated for 30 min. The sample was recovered with a magnet, washed twice with methanol, and then redispersed in a mixture containing 4 mL of methanol, 32 mg of Zn(NO 3 ) 2 ·6H 2 O and 82 mg of 2-MI. The suspension was sonicated for 20 min until homogeneous. Finally, the precipitate was collected, washed several times with methanol and water, and redispersed in 0.6 mL of deionized water.

[0011] Preferably, the step S3 includes: First, dissolve 25 mg of PS 45 -PVP 44 -PEO 113 in 1 mL of tetrahydrofuran (THF), and rapidly add 4 mL of distilled water to the above THF solution under stirring. The mixture was stirred for 15 min and then dialyzed with water. Add 0.2 mL of the Zn(CH 3 COO) 2 ·2H 2 O solution dissolved in the micelles to 0.3 mL of an aqueous solution containing Fe 3 O 4 @ZIF-8, and vigorously mix the suspension for 30 min. Then add the solution containing 80 mg of 2-MI to 1.5 mL of the micelle solution, rapidly mix it with the above suspension under ultrasound, and stir at 15 °C for 1 h. Recover the product with a magnet and then wash it with N,N-dimethylformamide (DMF). To remove the template PS 45 -PVP 44 -PEO 113 , the synthesized sample was soaked in ethanol at 60 °C for 2 d. Finally, the product was dried overnight under vacuum at 60 °C.

[0012] Preferably, in step S3, since the Fe 3 O 4 core has excellent magnetic separation ability, while the HMZIF-8 shell has large pore size and high pore space, Fe 3 O 4 @HMZIF-8 is expected to be used as a solid carrier for creating immunoassays in mesopores.

[0013] Preferably, the mesopore aperture of Fe 3 O 4 @HMZIF-8 is adjustable. In a preferred embodiment, the aperture is 43 nm.

[0014] According to the second aspect of the present invention, there is provided a mesopore immunoassay analysis model constructed according to the above preparation method, with the HMZIF-8 shell covering the capture antibody as a solid carrier, and superparamagnetic Fe3 O 4 The NPs core enables efficient recycling.

[0015] This invention is based on the synthesized Fe 3 O 4 @HMZIF-8-based chemiluminescence (CL) immunoassay (IMIA) was used to analyze immunoglobulin A (IgA), effectively verifying the above concept.

[0016] Preferably, the HMZIF-8 shell is used as a solid support to coat the capture antibody (Ab 1 ). A large amount of coated Ab 1 can effectively capture IgA in the three-dimensional mesopores of Fe 3 O 4 @HMZIF-8 and bind with the biotin-labeled secondary antibody (Ab 2 ) to form a sandwich system. Subsequently, streptavidin-labeled alkaline phosphatase (ALP) is added and tightly binds to the biotin-labeled Ab 2 . Finally, the added luminescent substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane (AMPPD) is converted into a strong and persistent luminescent body under the catalysis of ALP.

[0017] Preferably, the coating capacity of Ab 1 can be as high as 247 mg g -1 .

[0018] Preferably, the CL intensity increases linearly with the logarithm of the IgA concentration, from 10 2 -10 5 fM (16 pg mL -1 -16 ng mL -1 ), and the LOD is about 56 fM (9 pg mL -1 ).

[0019] According to the third aspect of this invention, there is also provided an application of an IMIA based on the above Fe 3 O 4 @HMZIF-8. The chemiluminescence (CL) intensity is positively correlated with the concentration of cardiac troponin I (cTnI). The concentration of cTnI can be quantified by detecting the CL intensity. cTnI is also the most valuable biomarker for diagnosing acute myocardial infarction. As the duration of myocardial injury prolongs, the level of cTnI in human blood will continuously increase. Accordingly, the chemiluminescence IMIA model developed based on Fe 3 O 4 @HMZIF-8 can achieve specific detection of cTnI.

[0020] Preferably, the invented IMIA model shows specificity in the detection of cTnI. Only the presence of cTnI can cause a strong CL intensity, while the luminescence intensity of its possible co-existing interfering substances shows little change.

[0021] Preferably, in the concentration range of 500 - 10 5 fM for the cTnI solution, the IMIA model is used to perform a luminescence response to cTnI, and a linear fitting analysis is carried out on the logarithm of the CL intensity and the cTnI concentration. Accordingly, the chemiluminescence IMIA model developed based on Fe 3 O 4 @HMZIF-8 can be used for the quantitative analysis of cTnI.

[0022] Preferably, the detection limit of cTnI is 366 fM.

[0023] Accordingly, the IMIA of the present invention can be used for clinical detection applications, and can directly quantitatively detect the content of cTnI in actual serum, quickly screen and diagnose patients with acute myocardial infarction. Among them, different concentrations of cTnI are added to the serum samples of healthy people to draw a calibration curve. Through the standard curve, the CL intensity is converted into the cTnI concentration to realize the judgment of the stage of patients with acute myocardial infarction in clinical serum.

[0024] The present invention prepares the Fe 3 O 4 NPs core by a solvothermal method; the outer surface of the Fe 3 O 4 NPs is modified with PVP, and PVP can strongly coordinate with Zn 2+ ions to generate sufficient interactions, solving the phase separation problem; Fe 3 O 4 @HMZIF-8 is prepared by a soft-template directed self-assembly and constrained growth strategy. With this unique structure, IMIA is realized, that is, the HMZIF-8 shell serves as a solid carrier to cover the capture antibody, and the superparamagnetic Fe 3 O 4 NPs core realizes efficient recycling. The three-dimensional large mesopores in this structure not only significantly improve the density of the capture antibody and the antibody-antigen binding efficiency, but also provide sufficient space for all bound biomacromolecules to integrate them into a mesoporous channel. At the same time, the involved biomacromolecules are encapsulated in Fe 3 O 4In the mesoporous channels of @HMZIF-8, it is beneficial to maintain its natural conformation and protect it from the harsh external environment. The developed IMIA can accurately quantify the slight fluctuations in the concentration of cTnI in serum samples at different stages after the onset of acute myocardial infarction patients. Compared with the traditional plate ELISA, the LOD is increased by more than 100 times. This IMIA model may open up a new way to achieve sensitive immunoassay and is expected to be widely used in the early monitoring and diagnosis of various diseases.

[0025] In summary, according to the method of the present invention, an immunoassay analysis model is constructed. By means of the large mesoporous channels to provide a stable microenvironment to maintain the recognition ability of the capture antibody, the sensitivity of the immunoassay is significantly improved. It can accurately quantify the content of low-concentration cTnI in serum, and the luminescence quantitative detection of cTnI in complex biological samples is not interfered. By changing the type of the primary capture antibody of the IMIA, a new nano-platform for accurate diagnosis of different diseases is obtained. Based on the efficient coverage of high-concentration antibodies in the solid carrier, portable, non-destructive and sensitive detection of ultra-low abundance biomarkers can be achieved. The immunoassay analysis model constructed according to the method of the present invention also has broad application potential in the early monitoring and diagnosis of various diseases. Brief Description of the Drawings

[0026] Figure 1 is a schematic diagram of the preparation process of IMIA based on Fe 3 O 4 @HMZIF-8 according to the present invention;

[0027] Figure 2 is the transmission electron microscope image of Fe 3 O 4 NPs (a) and the transmission electron microscope image of Fe 3 O 4 @ZIF-8 (b) according to the present invention;

[0028] Figure 3 is the transmission electron microscope image of Fe 3 O 4 @HMZIF-8 at reaction 5 min (a) and reaction 15 min (b) according to the present invention;

[0029] Figure 4 The transmission electron microscope (a) and scanning electron microscope (b) images of Fe 3 O 4 @HMZIF-8 according to the present invention;

[0030] Figure 5 is ZIF-8, uncoated Fe 3 O 4 NPs and Fe coated with HMZIFs3 O 4 XRD pattern of NPs;

[0031] Figure 6 is Fe according to the present invention 3 O 4 @HMZIF-8's N 2 adsorption isotherm and its corresponding BJH pore size distribution diagram;

[0032] Figure 7 is Fe according to the present invention 3 O 4 magnetization and demagnetization curves of Fe

[0033] Figure 8 is Fe according to the present invention 3 O 4 @HMZIF-8, HMZIF-8 and Fe 3 O 4 IR spectra of NPs;

[0034] Figure 9 is ZIF-8 synthesized without a template, Fe 3 O 4 @HMZIF-8 after removing the template agent and the template agent PS 45 -PVP 44 -PEO 113 IR spectra of;

[0035] Figure 10 is Fe according to the present invention 3 O 4 @HMZIF-8, HMZIF-8 and Fe 3 O 4 TG curves of NPs;

[0036] Figure 11 is at (1) Ab 1 , (2) BSA, (3) Fe 3 O 4 @HMZIF-8, (4) Fe 3 O 4 @Ab 1 / HMZIF-8 and (5) Fe 3 O 4 @BSA / Ab 1 Zeta potential under the condition that the pH value of / HMZIF-8 is 7.4;

[0037] Figure 12 is Fe according to the present invention 3 O 4@HMZIF-8 Coated Ab 1 XRD pattern after;

[0038] Figure 13 is Fe according to the present invention 3 O 4 @HMZIF-8 Coated Ab 1 Transmission electron microscopy (a) and scanning electron microscopy (b) images after;

[0039] Figure 14 is Fe according to the present invention 3 O 4 @HMZIF-8 Coated Ab 1 N after 2 Adsorption isotherm (a) and corresponding BJH pore size distribution plot (b);

[0040] Figure 15 is the standard fitting curve of absorbance vs. concentration of Ab 1 in PBS buffer;

[0041] Figure 16 is the plot of Fe 3 O 4 @Ab 1 / HMZIF-8 concentration in optimized CL IMIA;

[0042] Figure 17 is the plot of CL intensity vs. IgA concentration for three IA types;

[0043] Figure 18 is the standard curve for IMIA determination of IgA based on Fe 3 O 4 @Ab 1 / HMZIF-8 according to the present invention;

[0044] Figure 19 is the standard curve for IgA determination by conventional microplate-based CL IA;

[0045] Figure 20 is Fe 3 O 4 @Ab 1 / ZIF-8 CL IA determination of IgA standard curve;

[0046] Figure 21 is the specificity test plot for (1) ALP, (2) IgG, (3) PSA, (4) cTnI, (5) CEA, (6) AFP and (7) IgA by IMIA according to the present invention;

[0047] Figure 22 is Fe according to the present invention 3 O4 @Ab 1 / Anti-interference test diagrams of IMIA of HMZIF-8 against (1) IgA+ALP, (2) IgA+IgG, (3) IgA+PSA, (4) IgA+cTnI, (5) IgA+CEA, (6) IgA+AFP, (7) IgA;

[0048] Figure 23 is Ab 1 (1) and Fe 3 O 4 @Ab 1 / Activity change diagrams of HMZIF-8(2) after being soaked in different organic solutions for 7 days;

[0049] Figure 24 is Ab 1 (1) and Fe 3 O 4 @Ab 1 / Activity change diagrams of HMZIF-8(2) after being soaked in different alkaline aqueous solutions for 7 days;

[0050] Figure 25 is Fe 3 O 4 @Ab 1 / Diagrams of the relationship between CL intensity and IgA concentration of HMZIF-8 under different conditions, including freshly obtained, after being stored at 37 °C and 4 °C for 7 days;

[0051] Figure 26 is based on Fe 3 O 4 @Ab 1 / Standard curve for determining IgA by IMIA of HMZIF-8 after being stored at 37 °C for 7 days;

[0052] Figure 27 is based on Fe 3 O 4 @Ab 1 / Standard curve for determining IgA by IMIA of HMZIF-8 after being stored at 4 °C for 7 days;

[0053] Figure 28 is according to the present invention based on Fe 3 O 4 @Diagrams of the relationship between CL intensity and cTnI concentration determined by IMIA of HMZIF-8 according to the present invention;

[0054] Figure 29 is according to the present invention based on Fe 3 O 4 @Standard curve for determining cTnI concentration by CL intensity determined by IMIA of HMZIF-8 according to the present invention;

[0055] Figure 30 is the repeatability investigation of IMIA based on Fe 3 O 4 @HMZIF-8 for detecting 7 samples of the same concentration of cTnI (10 5 fM);

[0056] Figure 31 is the stability study of IMIA based on Fe 3 O 4 @HMZIF-8 for continuously detecting samples of the same concentration of cTnI (10 5 fM) for 6 days;

[0057] Figure 32 is the specificity test chart of IMIA based on Fe 3 O 4 @HMZIF-8 for (1) ALP, (2) IgG, (3) PSA, (4) IgA, (5) CEA, (6) AFP, and (7) cTnI;

[0058] Figure 33 is the anti-interference test chart of IMIA based on Fe 3 O 4 @HMZIF-8 for (1) cTnI + ALP, (2) cTnI + IgG, (3) cTnI + PSA, (4) cTnI + IgA, (5) cTnI + CEA, (6) cTnI + AFP, and (7) cTnI;

[0059] Figure 34 is the relationship chart of CL intensity in serum and cTnI concentration determined by IMIA based on Fe 3 O 4 @HMZIF-8;

[0060] Figure 35 is the calibration curve of CL intensity in serum and cTnI concentration determined by IMIA based on Fe 3 O 4 @HMZIF-8;

[0061] Figure 36 is the repeatability investigation of IMIA based on Fe 3 O 4 @HMZIF-8 for 5 identical samples of cTnI in serum with concentrations of 3×10 4 、5×10 4 and 10 5 fM respectively;

[0062] Figure 37 is the stability study of IMIA based on Fe 3 O 4 @HMZIF-8 for detecting cTnI in three samples containing 3×10 4 、5×10 4 and 10 5 fM in serum;

[0063] Figure 38 is the anti-interference test diagram of IMIA based on Fe 3 O 4 @HMZIF-8 for (1) IgA + cTnI, (2) IgG + cTnI, (3) PSA + cTnI, (4) CEA + cTnI, (5) cTnI in serum;

[0064] Figure 39 is the specificity test diagram of IMIA based on Fe 3 O 4 @HMZIF-8 for (1) IgA, (2) IgG, (3) PSA, (4) CEA and (5) cTnI in serum;

[0065] Figure 40 is the CL intensity of IMIA based on Fe 3 O 4 @HMZIF-8 for detecting cTnI concentration in serum samples of acute myocardial infarction patients 2 - 3 hours, 3 - 12 hours and 12 - 24 hours after symptom onset and healthy individuals. Detailed implementation manners

[0066] The following provides the preferred embodiments of the present invention in conjunction with the accompanying drawings and describes them in detail.

[0067] As Figure 1 shown, the preparation method of Fe 3 O 4 @HMZIF-8 according to the present invention includes the steps: Synthesize Fe 3 O 4 NPs by solvothermal method; then coat the outer surface of Fe 3 O 4 NPs with PVP polymer to promote the epitaxial growth of ZIF-8 on the surface of magnetic NPs, obtaining Fe 3 O 4 @ZIF-8; Through the soft-template-directed self-assembly and constrained growth strategy, and introducing a hydrophobic pore swelling agent, obtain magnetic Fe 3 O 4 @HMZIF-8 NPs, whose core is Fe3 O 4 NPs, with HMZIF-8 as the shell layer.

[0068] Example 1

[0069] 1.1Fe 3 O 4 Synthesis of NPs

[0070] At room temperature, 1.3 g of FeCl 3 ·6H 2 O and 0.5 g of sodium citrate were dissolved in 40 mL of ethylene glycol. Then, 1.2 g of CH 3 COONa was added and stirred for 30 min. The mixture was then transferred and sealed in a stainless-steel autoclave lined with polytetrafluoroethylene. The autoclave was heated at 200 °C for 8 h and then cooled to room temperature. Finally, it was washed several times with ethanol and deionized water. The product was dried overnight under vacuum at 60 °C.

[0071] 1.2Fe 3 O 4 Synthesis of @ZIF-8 Metal-Organic Framework

[0072] At room temperature, 10 mg of Fe 3 O 4 NPs were added to 2.4 mL of methanol containing 0.16 g of PVP and sonicated for 30 min. The sample was recovered with a magnet, washed twice with methanol, and redispersed in a mixture containing 4 mL of methanol, 32 mg of Zn(NO 3 ) 2 ·6H 2 O and 82 mg of 2-MI. The synthesized suspension was sonicated for 20 min until evenly dispersed. Finally, the precipitate was collected, washed several times with methanol and water, and then redispersed in 0.6 mL of deionized water to obtain an aqueous solution of Fe 3 O 4 @ZIF-8.

[0073] 1.3 On Fe 3 O 4 @ZIF-8, grow hierarchical mesoporous ZIFs (Fe 3 O 4 @HMZIF-8)

[0074] First, 25 mg of PS 45 -PVP 44 -PEO 113 was dissolved in 1 mL of tetrahydrofuran (THF). After stirring, 4 mL of distilled water was quickly added to the above THF solution; the mixture was stirred for 15 min and then dialyzed with water; then, 0.2 mL of a solution of Zn(CH 3 COO) 2 ·2H 2 O dissolved in the micelles was added to a solution containing Fe 3 O 4In 0.3 mL of aqueous solution of ZIF-8, the suspension was then vigorously mixed for 30 min; the solution containing 80 mg of 2-MI was added to 1.5 mL of micellar solution, ultrasonically mixed, and stirred at 15 °C for 1 h. The product was recovered with a magnet and then washed with DMF. To remove the template agent PS 45 -PVP 44 -PEO 113 , the synthesized sample was soaked in ethanol at 60 °C for 2 d. Finally, the product was dried in vacuo at 60 °C overnight to obtain Fe 3 O 4 @HMZIF-8.

[0075] Figure 2 where a in 3 O 4 is the transmission electron microscopy image of the synthesized Fe 3 O 4 NPs. The uncoated Fe Figure 2 where b in 3 O 4 is the transmission electron microscopy image of Fe Figure 3 is the transmission electron microscopy image of Fe 3 O 4 @HMZIF-8 monitored with reaction time. The reaction times are (a) 5 min and (b) 15 min. Mesoporous nuclei are formed in the initial stage of the reaction. Figure 4 is the (a) transmission electron microscopy and (b) scanning electron microscopy images of Fe 3 O 4 @HMZIF-8, showing a typical core-shell structure, regular rhombic dodecahedron. The thickness of the mesoporous shell is about 200 nm, while the average particle size of Fe 3 O 4 @HMZIF-8 is about 500 nm and has open mesopores on the surface. Figure 5 is the XRD patterns of ZIF-8, uncoated Fe 3 O 4 NPs and HMZIFs-coated Fe 3 O 4 NPs. The XRD pattern of the synthesized Fe 3 O 4 @HMZIF-8 matches well with those of Fe 3 O 4 NPs and ZIF-8. Due to the shielding effect of the HMZIF-8 shell, the peak intensity corresponding to Fe 3 O 4 @HMZIF-8 is slightly weaker than that of Fe 3 O 4 NPs.Figure 6 is Fe 3 O 4 N adsorption isotherm of Fe 2 @HMZIF-8 and its corresponding BJH pore size distribution diagram. Fe 3 O 4 N adsorption isotherm of @HMZIF-8 2 presents typical type I and type IV combined curves, accompanied by an H4-type hysteresis loop. The sharp rise at low relative pressure is related to the presence of intrinsic micropores, while the hysteresis loop between P / P 0 between 0.8 and 1.0 indicates the presence of mesopores in Fe 3 O 4 @HMZIF-8; according to the BJH pore size distribution diagram, the mesopore size distribution of Fe 3 O 4 @HMZIF-8 shows a peak centered at 43 nm. Figure 7 is the magnetization and demagnetization curves of Fe 3 O 4 @HMZIF-8, and the saturation magnetization value of the synthesized Fe 3 O 4 NPs is about 62.5 emu / g. Due to the increase of non-magnetic components, after the growth of HMZIF-8, the value of the obtained Fe 3 O 4 @HMZIF-8 drops to about 12.5 emu / g.

[0076] Figure 8 is the infrared spectra of Fe 3 O 4 @HMZIF-8, HMZIF-8 and Fe 3 O 4 NPs. The Fourier transform infrared spectrum of Fe 3 O 4 NPs shows characteristic bands corresponding to the vibration of carboxyl groups (-COOH) at 1411 and 1618 cm -1 and a band from the Fe-O bond at 613 cm -1 , confirming the successful synthesis of the composite NPs. Figure 9 shows the infrared spectra of ZIF-8 synthesized without a template, Fe 3 O 4 @HMZIF-8 after removing the template agent, and the template agent PS 45 -PVP 44 -PEO 113 , indicating that almost all of the template in the mesoporous channels of Fe 3 O 4 @HMZIF-8 has been removed through the solvent extraction process.Figure 10 is Fe 3 O 4 @HMZIF-8, HMZIF-8, and Fe 3 O 4 NPs thermogravimetric profiles. Due to the loss of physically and chemically adsorbed water and citric acid ligands, in the range of 200 - 400 °C, the weight loss rate of Fe 3 O 4 NPs is approximately 9%, and compared with pure HMZIF-8, the decomposition temperature of Fe 3 O 4 @HMZIF-8 is relatively lower, which may be due to the gradual decomposition of citric acid ligands and functionalized PVP on the surface of Fe 3 O 4 NPs.

[0077] Given that Fe 3 O 4 NPs have excellent magnetic separation ability and the HMZIF-8 shell has large pore size and high pore space, Fe 3 O 4 @HMZIF-8 is expected to become a solid carrier to construct an in-pore immunolab. As a proof of concept, based on the synthesized Fe 3 O 4 @HMZIF-8, CL IMIA detected common IgA. First, a high density of Ab 1 was loaded into the three-dimensional mesopores of HMZIF-8 to capture a large amount of IgA, and then biotin-labeled Ab 2 was added to form a sandwich system. Streptavidin-labeled ALP was added. Due to the highly specific affinity between streptavidin and biotin, streptavidin binds tightly to biotin-labeled Ab 2 ; finally, the luminescent substrate AMPPD was added and converted into a luminescent body under the catalysis of ALP to detect the CL intensity.

[0078] Example 2

[0079] 2.1 Coating with primary antibody Ab 1 Obtaining Fe 3 O 4 @Ab 1 / HMZIF-8

[0080] Mix the buffer solution (pH = 7.4, PBS) of Ab 1 (475 μg, 95 μL) with an aqueous solution (780 μL) containing 1.75 mg of Fe 3 O 4 @HMZIF-8. Shake the synthesized mixture uniformly at room temperature for 1 h to generate Fe 3 O 4 @Ab 1 / HMZIF-8 was collected by magnetic separation, washed with a buffer solution (pH = 7.4, PBS), and redispersed in PBS. Ab 1 / Fe 3 O 4 The final concentration of @HMZIF-8 was set at 50 mg / L. Subsequently, 100 μL of the Ab 1 / Fe 3 O 4 @HMZIF-8 suspension was added to the bottom of each test tube to prepare for subsequent experiments.

[0081] 2.2 Based on Fe 3 O 4 @Ab 1 Detection of IgA by IMIA of Fe O @Ab / HMZIF-8

[0082] First, 1 mL (1% wt) of BSA solution was added to a centrifuge tube and incubated at 37 °C for 2 h to block the non-specific binding sites on the Fe 3 O 4 @Ab 1 / HMZIF-8 structure. The unbound BSA was washed by magnetic separation with PBST (pH = 7.4, PBS buffer containing 0.1% wt Tween). Then, 1 mL of IgA protein standard solution (10 fM - 100 nM) was added to each test tube. After incubation at 37 °C for 1.5 h, the suspension was separated with a magnet and washed with PBST. Subsequently, 100 μL of biotin-labeled Ab 2 solution was added to the test tubes, incubated at 37 °C for 30 min, and washed with PBST. Then, 150 μL of streptavidin-labeled ALP solution (1 nM) was added and incubated at 37 °C for 30 min. It was washed with PBST and transferred to a 96-well plate. Subsequently, a mixed solution of AMPPD (15 μL, 5 mM) / MgCl 2 ·6H 2 O (15 μL, 100 mM) / 2-amino-2-methyl-1-propanol buffer (120 μL, pH = 9.5, 200 mM) was added to each well. The plate was kept at 37 °C for 15 min. The CL signal was recorded by a multi-functional microplate reader.

[0083] Figure 11 was in (1) Ab 1 、(2) BSA、(3) Fe 3 O 4 @HMZIF-8、(4) Fe 3 O 4 @Ab 1 / HMZIF-8 and (5) Fe 3 O 4 @BSA / Ab 1Zeta potential of Fe 3 O 4 @HMZIF-8 at pH 7.4. The Zeta potential of Fe 1 @HMZIF-8 changed from 16.2 ± 0.7 mV to 10.5 ± 0.9 mV after coating with Ab 1 and further decreased to -1.7 ± 0.8 mV after blocking with BSA. The change in Zeta potential indicated that Ab 3 O 4 might be successfully immobilized in the mesopores of Fe Figure 12 is the XRD pattern of Fe 1 O 3 @HMZIF-8 after coating with Ab 4 . Similar to the XRD pattern of the original Fe 3 O 4 @HMZIF-8, it confirmed that the introduction of Ab 1 did not reduce the crystallinity of the mesoporous walls. Figure 13 is the (a) transmission electron microscopy and (b) scanning electron microscopy images of Fe 1 O 3 @HMZIF-8 after coating with Ab 4 . The transmission electron microscopy image showed that open pores remained on the surface of Fe 3 O 4 @Ab 1 / HMZIF-8, indicating that the antibody did not block the mesoporous windows on HMZIF-8. The scanning image further demonstrated that the connected mesoporous channels and morphology remained intact after coating with Ab 1 . Figure 14 is the N 3 O 4 adsorption isotherm (a) and the corresponding BJH pore size distribution map (b) of Fe 1 O 2 @HMZIF-8 after coating with Ab 3 O 4 . The porosity of Fe

[0084] The following Table 1 gives the pore structure parameters of the synthesized Fe 3 O 4 @HMZIF-8 before and after coating with Ab 1 .

[0085] Table 1

[0086] Fe 3 O 4Ab in the mesopores of HMZIF-8 1 The quantity and density of Figure 15 Ab 1 are crucial for the sensitive detection of target analytes. Standard fitting curves of the absorbance of Ab 1 versus the concentration in PBS were obtained. Standard curve method was used to prepare Ab 1 standard solutions in PBS to analyze the encapsulation ability of Ab 3 in HMZIFs. Using a similar method, linear fitting of the absorbance at 280 nm was performed to plot the standard curve. The encapsulation ability of Ab 4 in Fe 1 O -1 @HMZIF-8 with a mesopore size of 43 nm was calculated to be approximately 247 mg g 3 O 4 @Ab 1 / HMZIF-8 greatly affects the sensitivity and linear range of IA. When introducing more Ab 1 , the sensitivity of IMIA increased, but the intensity of the blank signal also increased significantly. To balance the signal and noise, subsequent IA with a concentration of 50 ppm of Fe 3 O 4 @Ab 1 / HMZIF-8 was optimized. Figure 16 Shows the optimization of the concentration of Fe 3 O 4 @Ab 1 / HMZIF-8 in CL IMIA. Figure 17 Is a graph of the CL intensity of three IA types versus the IgA concentration. Under the above conditions, compared with the traditional microplate ELISA and IA based on Fe 3 O 4 @ZIF-8 in the same detection system, the currently developed IMIA detected the highest CL intensity. Figure 18 Is the calibration curve for the determination of IgA based on the developed IMIA. The CL intensity increased linearly with the logarithm of the IgA concentration, from 10 2 -10 5 fM (16 pg mL -1 -16 ng mL -1 ). Figure 19 Is the standard curve for the determination of IgA by conventional microplate-based CL IA; Figure 20 Is Fe 3 O 4 @Ab 1 / ZIF-8 CLIA standard curve for IgA determination, demonstrating a significant improvement in the LOD of the IMIA of the present invention compared to Fe 3 O 4 @ZIF-8 (LOD = 350 fM, 56 pg mL -1 ) and microplate IAs (LOD = 6 pM, 960 pg mL -1 ). The LOD was improved by approximately 6-fold and 107-fold, respectively. By introducing common antigens such as alpha-fetoprotein (AFP), immunoglobulin (IgG), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), cTnI, and ALP as interferents, the cross-reactivity of the developed IMIA was evaluated. Figure 21 Showing the specific recognition of the invented Fe 3 O 4 @Ab 1 / HMZIF-8-based IMIA for the detection of (1) ALP, (2) IgG, (3) PSA, (4) cTnI, (5) CEA, (6) AFP, and (7) IgA, the responses to all interfering biomolecules were negligible, while obvious CL signals were observed after the introduction of IgA. Figure 22 Is the anti-interference test of the prepared Fe 3 O 4 @Ab 1 / HMZIF-8-based IMIA for (1) IgA + ALP, (2) IgA + IgG, (3) IgA + PSA, (4) IgA + cTnI, (5) IgA + CEA, (6) IgA + AFP, (7) IgA. When these interferents coexisted in the IgA-containing detection system, there was no obvious effect on the sensing reaction, which confirmed the specific reaction of the IMIA to IgA. Figure 23 Shows the activity of Ab 1 (1) and Fe 3 O 4 @Ab 1 / HMZIF-8 (2) after being soaked in different organic solutions for 7 days. After exposure in DMF, dimethyl sulfoxide (DMSO), and ethanol (EtOH), the activity of Fe 3 O 4 @HMZIF-8-protected antibody remained above 75%. In contrast, the free antibody treated with EtOH only retained about 50% of its activity, while the activity of the free antibody exposed to DMSO and DMF decreased to 22%. Similarly, Figure 24 Shows the activity of Ab 1 (1) and Fe 3 O 4 @Ab 1 / HMZIF-8(2) activity after soaking in different alkaline aqueous solutions for 7 days. The results show that after treatment with alkaline aqueous solutions, the activity loss of Fe 3 O 4 @HMZIF-8-protected antibodies is negligible, while the activity of free antibodies is reduced by 30%. In addition, Figure 25 shows the activity of the prepared Fe 3 O 4 @Ab 1 / HMZIF-8 under different conditions, including freshly prepared and measured immediately, stored at 37 °C and 4 °C for 7 days; Figure 26 is the standard curve for IgA determination based on Fe 3 O 4 @Ab 1 / HMZIF-8 after storing at 37 °C for 7 days under the IMIA condition; Figure 27 is the standard curve for IgA determination based on Fe 3 O 4 @Ab 1 / HMZIF-8 after storing at 4 °C for 7 days under the CLIMIA condition. It is demonstrated that antibody-coated Fe 3 O 4 @HMZIF-8 stored at 37 °C and 4 °C for 7 days has a detection range comparable to that of freshly prepared samples, with LODs of 75 and 93 fM (12 and 15 pg mL -1 ), respectively.

[0087] 2.3 Based on Fe 3 O 4 Immunoassay of cTnI by IMIA of Fe @HMZIF-8

[0088] The detection procedure is similar to that of the above-mentioned IgA detection. cTnI is used as a biomarker for the diagnosis of acute myocardial infarction. Using the invented IMIA, different amounts of cTnI are added to the sensing system, and cTnI is quantified by detecting the CL intensity. Figure 28 shows the relationship diagram between the CL intensity and the cTnI concentration, and Figure 29 shows the corresponding fitting curve for cTnI determination. In the dynamic concentration range of 500 - 10 5 fM (30 - 6000 pg mL -1 ), there is a good linear correlation between the CL intensity and the logarithm of the cTnI concentration, and the calculated LOD is approximately 366 fM (22 pg mL -1 ).

[0089] Table 2 below compares the sensing properties of the developed Fe 3 O 4 @HMZIF-8-based IMIA with other reported IAs.

[0090] Table 2

[0091] Figure 30 is a repeatability investigation for detecting 7 samples of the same concentration of cTnI (10 5 fM); Figure 31 is a stability study for detecting samples of the same concentration of cTnI (10 5 fM) continuously for 6 days. To evaluate its repeatability and stability, we detected 7 identical cTnI solutions under optimized conditions using the proposed IMIA, showing stable CL intensity with a relative standard deviation (RSD) of 6.4%. Detecting the same sample continuously for 6 days can also maintain stable CL intensity with an RSD of 7.8%. Figure 32 is the specificity test of IMIA based on Fe 3 O 4 @HMZIF-8 for (1) ALP, (2) IgG, (3) PSA, (4) IgA, (5) CEA, (6) AFP, and (7) cTnI. The results show that when the IMIA system is exposed to interfering biomarkers, the observed CL signal is negligible, while different concentrations of the target cTnI result in significant changes in CL intensity. Figure 33 is the anti-interference test of IMIA based on Fe 3 O 4 @HMZIF-8 for (1) cTnI + ALP, (2) cTnI + IgG, (3) cTnI + PSA, (4) cTnI + IgA, (5) cTnI + CEA, (6) cTnI + AFP, and (7) cTnI. When the system is simultaneously added with the target cTnI and possible co-existing interferents, the CL intensity does not increase significantly compared to that of cTnI alone.

[0092] Application Example 1

[0093] 3.1 Based on Fe 3 O 4 The IMIA of @HMZIF-8 for the quantitative detection of cTnI in the clinical blood of patients with acute myocardial infarction

[0094] The detailed detection steps are similar to those for cTnI detection, except that diluted human serum samples are used instead of the cTnI standard solution. Real serum samples are diluted with PBS (pH = 7.4). The serum samples were donated by Jiashan First People's Hospital and include healthy individuals and patients with acute myocardial infarction (AMI) at different stages. First, different concentrations of cTnI were added to the serum matrix of healthy individuals using the standard addition method, and a calibration curve was plotted, observing a good linear correlation between CL intensity and cTnI level.

[0095] Figure 34 and 35Shows the relationship curve between CL intensity and cTnI concentration and the corresponding fitting curve of the developed IMIA for the determination of cTnI in serum. The developed IMIA can detect cTnI in the linear range of 500 fM to 10 5 fM (30 - 6000 pg mL -1 ). Its LOD is basically the same as that of non - serum matrix, which confirms its ability to detect biomarkers in real serum. Figure 36 Is the repeatability investigation of detecting 5 identical cTnI samples with concentrations of 3×10 4 , 5×10 4 and 10 5 fM in serum using the proposed IMIA; Figure 37 Is the stability study for 5 consecutive days of detecting three cTnI samples with concentrations of 3×10 4 , 5×10 4 and 10 5 fM in serum using the proposed IMIA. By detecting three spiked serum samples with cTnI concentrations of 3×10 4 , 5×10 4 and 10 5 fM, the accuracy and precision of the IMIA were evaluated within - batch and between - batches. The within - batch recovery rate was 96.1% - 105.5%, and the coefficient of variation (CV) was 2.5% - 5.5%. While the between - batch recovery rate was 103.1% - 104.4%, and the CV was 5.1% - 7.2%, indicating acceptable accuracy and repeatability. The results in Table 3 below verified the recovery test results of the IMIA for the determination of cTnI in serum samples.

[0096] Table 3

[0097] Figure 38 Shows the anti - interference performance of the Fe 3 O 4 @HMZIF - 8 - based IMIA for (1) IgA + cTnI, (2) IgG + cTnI, (3) PSA + cTnI, (4) CEA + cTnI, (5) cTnI in serum. Figure 39 Shows the selective detection performance of the prepared Fe 3 O 4 @HMZIF - 8 - based IMIA for (1) IgA, (2) IgG, (3) PSA, (4) CEA and (5) cTnI in serum. Experiments show that whether there are potential interfering proteins or not, only the presence of the target cTnI can cause a strong change in CL, which confirms the specificity of the IMIA for the detection of cTnI in serum samples.

[0098] Figure 40 The CL intensities for determining the cTnI concentrations in serum samples within 2 - 3 h, 3 - 12 h, and 12 - 24 h after symptom onset in AMI patients and in healthy individuals using IMIA are shown. The results indicate that compared with healthy people, serum samples from AMI patients result in a significant increase in the CL intensity of the IMIA detection system, and the CL intensity also varies at different stages after symptom onset. By converting the CL intensity into biomarker concentration, in patient samples collected 2 - 3 h after symptom onset, the cTnI level is approximately 0.22 ng / mL -1 , while for patient samples within 3 - 12 and 12 - 24 h after symptom onset, they are approximately 0.98 - 1.94 and 5.50 - 7.11 ng / mL respectively -1 . Using Fe 3 O 4 @HMZIF - 8 immunosensor, the detected values are consistent with those measured using a commercial ELISA kit. This clearly shows that the invented IMIA can provide a reliable monitoring platform for detecting low - abundance cTnI biomarkers in AMI patients. Table 4 below uses an ELISA kit and the developed Fe 3 O 4 @HMZIF - 8 immunosensor to compare the cTnI detected in human serum samples, verifying the sensitive immunoassay of IMIA.

[0099] Table 4

[0100] The present invention utilizes a soft - template - directed self - assembly and constrained - growth mechanism to enable the continuous crystallization of HMZIF - 8 on the surface of Fe 3 O 4 @ZIF - 8 to form a mesoporous shell layer, obtaining a magnetic core - shell solid support. The mesoporous ZIF shell can serve as an immunolab, while the magnetic Fe 3 O 4 NPs core helps with rapid separation, and based on this, IMIA is constructed. This model can accurately and quantitatively measure the slight fluctuations in the cTnI concentration in serum samples at different stages after the onset of acute myocardial infarction. Compared with traditional ELISA, the detection limit is increased by more than 100 times. This method is fast, efficient, and specific, opening up a new way for realizing sensitive AI and having broad prospects in the early detection and diagnosis of various diseases.

[0101] The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of protection of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a mesoporous immunoassay analysis model based on core-shell structured magnetic hierarchical porous ZIFs, characterized in that: The preparation method comprises the following steps: S1, synthesis of Fe3O4 NPs by solvothermal method; S2, coating the outer surface of Fe3O4 NPs with polyvinyl pyrrolidone polymer to promote the epitaxial growth of ZIF-8 on the surface of magnetic NPs to obtain Fe3O4@ZIF-8; S3, a superparamagnetic core-shell structure Fe3O4@HMZIF-8 was obtained through soft template-guided self-assembly and restricted growth strategy, whose core is Fe3O4 NPs and HMZIF-8 is used as the shell layer. This unique core-shell structure realizes the construction of immunoassay analysis model in mesoporous channels.

2. The preparation method according to claim 1, characterized in that: In step S2, the outer surface of Fe3O4 NPs is modified with polyvinyl pyrrolidone, wherein the hydrophilic PVP segment can react with Zn 2+ coordination, providing sufficient interactions to solve the phase separation problem and ensure the self-assembly of ZIF-8 on Fe3O4NPs.

3. The preparation method according to claim 1, characterized in that: The step S3 comprises: using PS 45 -PVP 44 -PEO 113 By anchoring metal ions for the template agent and introducing a pore expander, HMZIF-8 continuously crystallizes on the Fe3O4@ZIF-8 surface in a micellar environment to form a mesoporous shell.

4. The preparation method according to claim 3, characterized in that: In step S3, PS 45 -PVP 44 -PEO 113 The unique core-shell corona structure of the micelles plays a structural guiding role, among which PS, PVP, and PEO play the roles of hydrophobic core, hydrophilic shell, and solubilizing corona, respectively. The strong coordination between the N atoms in the hydrophilic PVP block and the metal ions ensures that the ZIFs precursor is fixed around the micelles.

5. The preparation method according to claim 1, characterized in that: The mesopore size of the synthesized Fe3O4@HMZIF-8 is adjustable in the range of 25-50nm.

6. A mesoporous immunoassay analysis model obtained according to the preparation method according to any one of claims 1 to 5, characterized in that: The HMZIF-8 shell serves as a solid carrier to cover the capture antibody, and the superparamagnetic Fe3O4 NPs core achieves efficient recovery.

7. Use of the mesopore immunoassay analysis model according to claim 6 in detecting cardiac troponin I, characterized in that: The concentration of cardiac troponin I is quantified by detecting the chemiluminescence intensity, which is positively correlated with the cardiac troponin I concentration.

8. The use according to claim 7, characterized in that: The mesopore immunoassay analysis model is specific for the detection of cardiac troponin I.

9. The use according to claim 7, characterized in that: When cardiac troponin I is between 500-10 5 In the concentration range of fM, when cardiac troponin I is detected using the mesopore immunoassay analysis model, the chemiluminescence intensity and the logarithm of the cardiac troponin concentration show a good linear correlation.

10. The use according to claim 9, characterized in that: The detection limit of cardiac troponin I using the mesopore immunoassay analysis model was 366 fM.

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